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S Alberti

Publications and source records attributed to S Alberti.

At least 55 records · Page 3Linked to original sources
PubMed1996

High-efficiency expression gene cloning by flow cytometry.

Our goal was to develop a convenient and widely applicable procedure for gene cloning based on flow cytometry. To this purpose, we have developed an efficient protocol for DNA transfection and selection of rare transfectants. Transfection by calcium phosphate co-precipitation was extensively investigated. The use of specific batches of calcium chloride, of carrier DNA purified in guanidinium thiocyanate, and of plasmid DNA banded in cesium chloride proved crucial for high efficiency of transfection. Several tissue culture parameters were also found critical. With the optimized procedure we can transfect almost 100% of the COS-7 cells with cDNA encoding cell surface antigens or green fluorescent protein. Moreover, we routinely obtain high average levels of expression. Efficient cell sorting in flow cytometry was achieved by subtracting the cell autofluorescence background, by displacing stained cells in the red dimension, and by combining fluorescein-conjugated primary and secondary antibodies. Efficient recovery of the transfected DNA constructs was obtained from 2500-3000 cells directly sorted in Hirt lysis buffer. Using the above protocol we have cloned by expression the gene encoding Trop-2, a cell surface glycoprotein expressed by human carcinomas.

Animals↗
PubMed1995

Cloning of the gene encoding Trop-2, a cell-surface glycoprotein expressed by human carcinomas.

We have cloned by expression the cDNA encoding Trop-2, a cell-surface glycoprotein expressed by most human carcinomas. Formal proof of the identity of the clone is the hybridization to DNA and RNA from genomic TROP2 transfectants. TROP2 is a single-copy gene in human cells, hybridizes to a single 1.8-kb mRNA from expressing sources and encodes a 35,709 Da type-1 transmembrane protein with a single transmembrane domain. TROP2 is essentially identical to GA733-1. Thus, we have proven that GA733-1, for which a protein product had not been identified, is a functional gene. TROP2 is also homologous to TROP1/KSA/GA733-2, confirming the serological similarities between the 2 molecules. The homology between the Trop-1 and Trop-2 peptides is clustered in 2 extracytoplasmic domains and in the transmembrane/cytoplasmic region. Twelve cysteines and a potential cytoplasmic tyrosine phosphorylation site are also conserved. Trop-1 and Trop-2 are homologous to serum IGF-II-binding proteins and appear as signal transducers. Thus, they likely represent novel cell-surface receptors and may play a role in regulating the growth of carcinoma cells. On the other hand, we have found no evidence for a role of Trop-2 and Trop-1 as homophilic adhesion molecules.

Amino Acid Sequence↗
PubMed1994

DNA methylation prevents the amplification of TROP1, a tumor-associated cell surface antigen gene.

We tested the hypothesis that different genes can have different abilities to be amplified after transfection under comparable selection conditions. DNA from human lymphoid or choriocarcinoma cell lines was transfected into L cells. Transfectants for CD5, CD8A, TROP1, and TROP2, genes expressed on lymphocytes or trophoblast and carcinomas, were selected by fluorescence-activated cell sorting. To select for amplification of the transfected gene we cloned twice by fluorescence-activated cell sorting the transfectants with the highest expression. We analyzed a total of 38 families (1768 clones) derived from the original transfectants. We then analyzed by Southern blotting the clones with the highest increase in surface expression and determined the copy number of each transfected gene. CD5, CD8A, and TROP2 were amplified with high frequency and progressively, whereas TROP1 essentially was not amplified at all. We examined the hypothesis that DNA methylation prevents the amplification of the TROP1 gene by treating JAR choriocarcinoma cells with 5-azacytidine to decrease DNA methylation. DNA extracted at different times after the treatment was used for transfection. When DNA that showed demethylation of the TROP1 gene was used, 16 Trop-1 transfectants were obtained and 6 of them were found to contain up to 40 copies of the TROP1 gene per haploid genome. Thus, we showed that transfectants obtained from a demethylated TROP1 gene were amplified efficiently and progressively. We propose that DNA methylation affects DNA amplification either by altering the recognition of methylated DNA sequences or by changing the conformation of the chromatin of methylated segments. We speculate that DNA methylation is a determinant of gene amplification in vivo, for example in tumor cells.

Animals↗
PubMed1994

Wild-type operator binding and altered cooperativity for inducer binding of lac repressor dimer mutant R3.

Substitution of the C-terminal leucine heptad repeat region of the normally tetrameric lactose repressor by the leucine heptad repeat dimerization domain of GCN4 protein resulted in cell extracts containing protein, designated R3, which behaved as a dimer based on gel retardation analysis of DNA binding (Alberti, S., Oehler, S., von Wilcken-Bergmann, B., and Müller-Hill, B. (1993) EMBO J. 12, 3227-3236). We have purified this R3 protein and characterized its properties in comparison with the wild-type repressor. R3 protein elutes from a molecular sieve with a Stokes radius characteristic of a dimer and a deduced molecular mass of 66 kDa. Unlike other dimeric repressors, produced by deletion or mutation in the leucine heptad repeat region, which display reduced apparent operator affinity, R3 binds to operator DNA sequences with wild-type equilibrium and kinetic properties. Although inducer affinity at neutral pH is similar for R3 and wild-type protein, at elevated pH the R3 protein undergoes a slightly smaller decrease in affinity and exhibits minimal cooperativity in sugar binding compared with the wild-type protein. Interestingly, in the presence of operator DNA, a state in which inducer binding to wild-type repressor is also of reduced affinity and slightly cooperative, R3 binding affinity is decreased to a greater extent, and the protein displays higher cooperativity than wild-type repressor. Consistent with inducer binding data in the presence of operator, the release of operator from R3 protein requires a higher sugar concentration than wild-type protein. These results are interpreted in the context of alterations involving the subunit interface which affect the allosteric behavior of the repressor protein.

Allosteric Regulation↗
PubMed1994

MRC OX19 recognizes the rat CD5 surface glycoprotein, but does not provide evidence for a population of CD5bright B cells.

To clone the rat CD5 gene we first produced two rat CD5 probes. The probes were obtained by polymerase chain reaction (PCR) on rat genomic DNA using primers designed on conserved regions between mouse and human CD5. The screening of a rat cDNA library at high stringency using these probes resulted in a 1.5-kb positive clone. The DNA sequence of this clone confirmed its CD5 nature, but the clone appeared to lack part of the 5' and part of the 3' end. These missing 5' and 3' ends were obtained by PCR on rat thymus RNA. By ligating these PCR products to the original 1.5-kb CDM8 clone, a full-length rat CD5 gene was constructed. The full-length clone showed high identity with mouse and human CD5; however, at the 5' site of the gene a region of 36 nucleotides is present which is not seen in either mouse or human CD5. We have evidence that this sequence is a normal constituent of the rat CD5 gene: first, it is in frame with the rest of the CD5 coding sequence; second, it does not contain a stop codon; and third, it is also present in the CD5 gene of other rat strains. We transfected the full-length CD5 construct in COS cells and demonstrated that indeed the CD5 protein is recognized by MRC OX19. Although we showed that CD5 mRNA is present in rat B cells, extensive flow cytometry analysis using MRC OX19 as antibody failed to detect B cells expressing significant levels of CD5 on their cell surface compared to other B cells in any tissue or cell suspension tested from a variety of rat strains. This is in contrast with the mouse where a distinct population of B cells (B-1a cells) can be found expressing more CD5 than the other B cells. Either B-1 cells are not present in rats or CD5 is not the right phenotypic marker for rat B-1 cells. It still remains to be investigated whether a population of B cells with functions similar to those of murine B-1 cells is present in rats.

Amino Acid Sequence↗
PubMed1994

In vivo targeting of integrin receptors in human skin xenografts by intravenously applied antibodies.

We examined whether systemically injected monoclonal antibodies (mAbs) directed to cell-surface glycoproteins of human keratinocytes reach their target antigens in xenograft transplants of normal human skin on SCID mice. The integrins alpha 6 beta 4, expressed in the basal cell layer of human epidermis, and glycoprotein T16 (gp 40/50), expressed in terminally differentiating keratinocytes of the stratum spinosum, were selected as targets. It was found that all injected mAbs selectively localized to their antigens and bound and saturated their targets even in the uppermost layers of the stratum malpighii. This could easily be monitored by direct immunofluorescence staining since SCID mice lack endogenous production of significant amounts of immunoglobulins. After a single injection, mAbs could still be detected at the target site after 14 days. Our results proved that heterologous immunoglobulins pass systemic capillary filters in this xenograft model and specifically bind to their target molecules. Thus, xenografted SCID mice provide a versatile model for studying cell-surface glycoprotein-mediated interactions by the use of functionally interfering antibodies under in vivo conditions in human skin.

Animals↗
PubMed1993

Biochemical and immunological characterization of the human carcinoma-associated antigen MH 99/KS 1/4.

We have characterized the 38-kDa transformation-associated membrane glycoprotein MH 99, whose expression is highly elevated in many epithelial malignancies. A spontaneous cleavage of MH 99 into a 32- and a 6-kDa chain in some carcinoma cell lines was recently shown. Sequence homologies to nidogen, a matrix-adhesion molecule, support the suggestion of a receptor-like function. In this study, we characterized biochemical and immunogenic aspects of MH 99. Transformed epithelial cell lines which do not spontaneously cleave MH 99 were exposed to 8 proteases with distinct specificities. Each of the enzymes produced similar specific fragmentation into chains of about 30 to 32 and 6 kDa, indicating a characteristic cleavage site of MH 99. The fragments were not distinguishable from those in carcinoma cells showing spontaneous cleavage of MH 99. The specific fragmentation depends on the localization in intact membranes and is not shared by other membrane proteins. N-glycosylation of MH 99 of about 4 kDa was exclusively found on the 32-kDa fragment. Characterization of antigenic epitopes was performed using 16 different monoclonal antibodies (MAbs). Only 2 independent determinants were found. One is located on the 32-kDa chain and is recognized only by the MM 104 MAb. The other 15 antibodies bind to a dominant epitope on the 6-kDa fragment which can be divided into 3 overlapping sub-epitopes. The unique features of MH 99 indicate that its immunogenic epitopes are mainly located at its 6-kDa chain, and support the suggestion of a transformation-associated cell-surface receptor which might be proteolytically regulated.

Animals↗
PubMed1993

T-cell-receptor engagement and tumor ICAM-1 up-regulation are required to by-pass low susceptibility of melanoma cells to autologous CTL-mediated lysis.

Tumor-specific and non-specific CD3+, TcR alpha beta+, CD8+ cytotoxic T-cell (CTL) clones, isolated from tumor-infiltrating lymphocytes (TIL) or peripheral blood lymphocytes (PBL) of a melanoma patient and allogeneic LAK cells, were used to investigate the requirements for bypassing the low lysability of some melanoma clones derived from an s.c. metastasis from which highly lysable clones were also obtained. Cytofluorimetric analysis showed that all melanoma clones expressed ICAM-1, although to different extents, reaching a 10-fold difference in fluorescence units, while HLA class-I antigens were similarly expressed. The differences in expression of ICAM-1 among tumor clones correlated with differences in lysability, by both specific and non-specific CTL, but were not large enough to affect lymphocyte-tumor conjugate formation. Cytokine- or gene-transfer-mediated up-regulation of ICAM-1 did not induce de novo lysis of ICAM-1low tumor cells; however, it markedly enhanced a low level of killing of the same cells by tumor-specific, TcR-dependent and HLA-restricted CTL clones but not by non-specific, TcR-independent effectors. In addition, lysis of melanoma clones by any effector was similarly inhibited by anti-ICAM-1 and anti-LFA-1 antibodies. This indicates that by-pass of low lysability of ICAM-1low melanoma clones by CTL clones, after ICAM-1 up-regulation, is possible only if simultaneous LFA-1 and TcR engagement takes place. In addition, these results suggest that the constitutive high level of expression of ICAM-1 on the subset of ICAM-1high melanoma cells must be only one of the factors contributing to the high lysability of these cells by any effector.

Cell Adhesion Molecules↗
PubMed1993

Genetic analysis of the leucine heptad repeats of Lac repressor: evidence for a 4-helical bundle.

Gel-filtration experiments indicate that a peptide (P2) composed of the basic region of GCN4 fused to the leucine heptad repeats of Lac repressor forms tetrameric aggregates. Gel-shift experiments were performed to determine the orientation of the helices in the tetrameric P2 aggregate. Sandwich-complex formation of peptide P2 with two DNA fragments containing two symmetrical CRE binding sites (5'-ATGACGTCAT-3') at a distance of 21 bp suggests antiparallel aggregation of the Lac leucine heptad repeats. Thus, we conclude that the leucine heptad repeats of Lac repressor have the ability to form homomeric 4-helical bundles with an antiparallel arrangement of the helices. This topology enables the two DNA fragments in the sandwich complexes to be held together by two tetramers of peptide P2. Replacement of the uncharged amino acids of the helical g and e positions of peptide P2 by the corresponding charged residues of GCN4 (peptide P4) results in a dimeric and parallel aggregation of the leucine heptad repeats, and consequently abolishes the potential to form sandwich structures. Similarly, a hybrid Lac repressor in which the GCN4 leucine zipper replaces the natural Lac leucine heptad repeats forms dimers only. It regains the ability to form tetramers when the charged amino acids in helical positions g and e are replaced by uncharged alanines.

Amino Acid Sequence↗
PubMed1992

Membrane association and shedding of the GPI-anchored Ca-MOv18 antigen in human ovary carcinoma cells.

The antigen recognized by the MOv18 MAb (Ca-MOv18) was recently shown to be a glycosylphosphatidylinositol (GPI)-linked protein. In this report we show that GPI-anchorage is not limited to IGROVI cells nor to other ovary carcinoma cell lines, but Ca-MOv18 was also found to be sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC) treatment on fresh ovarian cancer cells. Furthermore, we found a heterogeneous sensitivity of Ca-MOv18 to PI-PLC cleavage, not only among the different cells studied but also in different experiments performed on the same cell line, during extended periods of time in culture. Sensitivity to PI-PLC cleavage was determined by immunofluorescence on live cells and by double-determinant radioimmunoassay of the antigen released in the supernatant. The specificity of the PI-PLC cleavage was demonstrated as follows: (a) TX114 solubilized Ca-MOv18 shifts from the detergent to the aqueous phase after treatment with PI-PLC; (b) on membrane preparations, PI-PLC specifically released a fraction of the antigen, which is distinct from the weakly associated form released by high-salt treatment; (c) Ca-MOv18 from IGROVI expressed the cross-reacting determinant (CRD), which is characteristic of GPI-linked molecules. The absence of CRD expression on the spontaneously released protein and the possibility of artificially inducing antigen shedding during the resynthesis of Ca-MOv18 which follows bacterial PI-PLC treatment are interesting points which need to be further investigated in order to understand the physiology of the Ca-MOv18 tumor antigen.

Antibodies, Monoclonal↗
PubMed1992

Heterogeneous susceptibility of human melanoma clones to monocyte cytotoxicity: role of ICAM-1 defined by antibody blocking and gene transfer.

Five clones derived from the same human malignant melanoma lesion were studied for their susceptibility to killing by human monocytes activated by exposure to interferon (IFN)-gamma and lipopolysaccharide. Melanoma clones were heterogeneous in their susceptibility to human monocyte cytotoxicity, with one clone (2/21) exhibiting extremely low levels of lysis. The different levels of susceptibility to monocyte cytotoxicity were not accounted for by susceptibility or resistance to monokines [tumor necrosis factor (TNF), interleukin (IL)-1 and IL-6] because: (a) these effector molecules had little (TNF) or no (IL-1 and IL-6) cytolytic activity under these conditions; and (b) anti-TNF antibodies had marginal effects on cytotoxicity. Monocytes bound less to resistant than to susceptible melanoma cells. Monocyte-resistant 2/21 melanoma cells expressed substantially lower levels of ICAM-1 and VLA-4 than susceptible cells. Anti-CD18 and, to a lesser extent, anti-ICAM-1 mAb inhibited binding and cytotoxicity of human monocytes on malignant melanoma whereas anti-VLA-4 had no inhibitory action. Transfection of the ICAM-1 gene under the control of a constitutive promotor resulted in high levels of expression of ICAM-1 in 2/21 melanoma cells and, concomitantly, in augmented susceptibility to activated monocyte cytotoxicity. The augmented killing of ICAM-1 transfected 2/21 cells was inhibited by anti-ICAM-1 mAb. These results demonstrate that the CD18-ICAM-1 adhesion pathway can play an important role in the expression of human monocyte cytotoxicity on melanoma target cells and that heterogeneity in expression of ICAM-1 can underlie differences in susceptibility to tumoricidal activity.

Animals↗
PubMed1992

Biochemical characterization of Trop-2, a cell surface molecule expressed by human carcinomas: formal proof that the monoclonal antibodies T16 and MOv-16 recognize Trop-2.

Trop-2 is a cell surface structure recognized by the 162-46.2 mAb and expressed by most human carcinomas. Since the 162-46.2 mAb works poorly in immunoprecipitation, to characterize the structure of Trop-2 we searched for other mAbs directed against this molecule. Selection of candidates was performed by analyzing the characteristics of mAbs directed against epithelial cells and by comparing the staining pattern of each mAb with the one of the 162-46.2 on frozen sections of human epidermis. Two mAbs, T16 and MOv-16, were selected for further analysis. Formal proof that candidate mAbs reacted with Trop-2 was obtained by comparing their binding patterns to mouse L cells transfected with the Trop-2 gene by genomic DNA transfection and selected by FACS using the FITC-162-46.2 mAb. In immunofluorescence FACS analysis the FITC-T16 and FITC-MOv-16 mAbs specifically stained Trop-2 transfectants. The specificity of binding was confirmed by selective blocking of the staining by the respective unconjugated mAb. Interestingly, cross-blocking studies indicated that the 162-46.2, T16 and MOv-16 mAbs recognize the same epitope or closely spaced ones on the Trop-2 molecule. T16 and MOv-16 efficiently immunoprecipitate Trop-2 from Trop-2 transfectants and from the human cell line OVCA-432, indicating that it is a cell surface glycoprotein, with an apparent molecular weight of 57 kD in non-reducing conditions. A weaker band of 38 kD is often co-precipitated with the 57 kD form in an apparently specific manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1992

Molecular cloning, reconstruction and expression of the gene encoding the alpha-chain of the bovine CD8--definition of three peptide regions conserved across species.

We report the cloning of a cDNA encoding the alpha-chain of the bovine CD8 (BoCD8 alpha). A bovine thymus cDNA library was hybridized at low stringency with a human CD8 alpha cDNA clone. The first round of screening of 5 x 10(4) independent colonies yielded 12 clones containing incomplete BoCD8 alpha genes. Two further rounds of colony hybridization were conducted, each using as a probe the 5' fragment from the longest BoCD8 alpha clone previously isolated. The final screening yielded a clone containing a 2 kilobase (kb) insert. We mapped and sequenced the 2 kb BoCD8 alpha clone and compared it with the published sequences of the genes encoding the human, mouse and rat CD8 alpha. Sequence analysis confirmed that the clone under study encoded the BoCD8 alpha. The overall similarity of the BoCD8 alpha coding region with the human CD8 alpha coding sequence is 74.7% at the nucleotide level and 62.1% at the protein level. Lower levels of similarity are found with the mouse and rat CD8 alpha. Interestingly, three separate highly homologous regions are clearly defined at the peptide level in bovine versus human and mouse versus rat comparisons. Two of the regions are highly conserved among all species analysed, while the most 5' region is not. We speculate that the latter region may contain the binding site of CD8 alpha to the alpha 3 domain of major histocompatibility complex (MHC) class I molecules. Sequence analysis showed that the 2 kb BoCD8 alpha clone contains an incomplete coding region, i.e. lacks six bases corresponding to the first two amino acids of the leader region. To allow efficient translation and processing of the BoCD8 alpha gene, we constructed a chimeric gene containing the coding sequence of the BoCD8 alpha clone and synthetic sequences corresponding to the first two amino acids of the human CD8 alpha leader sequence. The chimeric gene was subcloned in the pKSV10 expression vector. The pKSV10-BoCD8 alpha construct is efficiently expressed both transiently in COS cells and stably in L cells, as determined by Northern blot and by FACS analysis, using the ILA-51 monoclonal antibody to BoCD8 alpha. The latter result formally proves that the ILA-51 antibody does indeed recognize the product of the BoCD8 alpha gene, as previously suggested on serological grounds.

Amino Acid Sequence↗